1. Technical Field
This present invention relates generally to methods of detecting and determining the identity, amount and activity of molecules with biological activity. More specifically, the present invention is concerned with a method of measuring the amount and activity of enzymes, enzyme inhibitors, lectins, receptors and other biologically active molecules via solid-phase assay techniques.
2. Background Art
Binder-ligand assays, such as immunoassays, are well known in the art and are used to quantitate the presence of a substance utilizing antibody based identification. (see Stites et al, Basic and Clinical Immunology, 8th edition, Appleton and Lange, pgs 170-176 for a review). However, immunoassays have two disadvantages. The first disadvantage is that they require an antibody raised against the substance of interest. In the second, if the substance being identified has biologically activity, for example an enzyme, the immunoassay cannot determine the level of activity of the substance, only its presence.
There are assays for enzyme activity, receptor function and the like, but it would be useful to have such assays utilizing solid-phase technology and accuracy with minimal preparation of components, i.e. no physical separation step, and that can be readily adapted to a routine assay of large number of samples.
As an example, enzymes play a key role in biochemical reactions. The determination of their activity is important in all fields related to biology such as medicine, food and pharmacy. The methods currently used for enzyme assays are mainly based on the formation of product from substrate following enzyme catalysis (Loround, 1981; Rossomando, 1990). Although these methods are the mainstream of much biological research there is also a need to not only determine the activity of an enzyme but also the quantity of enzyme present as well as its identity. This is particularly important for an enzyme for which its activity does not follow normal Michaeleus-Menton kinetics such as the allosteric enzymes or those that are activated by covalent modification.
In addition there is a need for the quantitative determination of the amount of enzyme inhibitor that may be present in a biological system. This is particularly important in the medical field where much attention has been directed towards the discovery of inhibitors of certain enzymes in AIDS research (Miller et al, 1989) or the development of inhibitors for the control of blood pressure (Ondetti et al., 1982) or the hydrolysis of antibiotics including penicillin (Cullman, 1990).
There is also a urgent demand for enzyme assays that can be automatized especially in the pharmaceutical industry as enzyme are usually used as a target for drug discovery. Thousands of chemical compounds must be screened for the search for new drugs. The development of a new method for the assay of enzymes that is amenable to high throughput screening and automation would not only greatly facilitate such screening but would also have many other applications and would result in markedly reduced costs. In addition to enzymes, highly automated, screening assays for measurements of receptors and lectins are also needed.
For example, there has been a continuing interest in the development of simple and reliable assay procedures for xcex2-glucanase, as this enzyme plays an important role in the depolymerization of barley xcex2-glucan in both the brewing and the poultry production industries. Several methods have been reported for this assay including viscometry (Bourne and Pierce, 1970), reducing sugar production (Denalt et al., 1978), radial gels diffusion (Edney et al., 1986; Martin and Bamforth, 1983) and the use of azo-barley glucan (McCleary and Shameer, 1987). The detection and the quantitation of enzyme activity in finished feeds by any method developed to date is technically challenging due to the requirement for high sensitivity and the complex nature of feed itself. The development of a highly sensitive photometric method will be welcomed particularly if this could lead to a high degree of assay automation. Microtitration using micro-titre plates and a microtitre plate reader would greatly facilitate such an assay.
There have been two approaches in this direction, one was the studies of Wirth and Wolf (1992) using a micro-plate calorimetric assay. The principle of this assay is the same as the azo-barley glucan method except the absorbency is read in microtitre plate wells. This procedure, as well as the original azo-barley glucan procedure, has the disadvantage of requiring a precipitant and a centrifuging step. It also does not have a high degree of sensitivity. Another approach has been to quantitate the amount of enzyme using the immunological properties of enzymes (Bxc3xchler, 1991; Rafael et al., 1995). The main drawback of this technique is its inability to assess the biological activity of a particular enzyme, as the immunoassay will estimate the amount of enzyme protein but not its biological activity. Also this assay would only be useful for enzyme from closely related species as antibodies tend to have high specificity. In a recent review Headon (1993) concluded that no suitable method has been reported that facilitates detection and quantitation of enzymes added to feed. This may in part be attributed to the lack of an assay that is able to detect the very low levels of enzymes that are usually added to feed.
Additionally, the availability of a simple, sensitive and efficient method for the assay of protease activity would be very useful for the recombinant protein industry to test intrinsic proteolytic activity, drug discovery to screen for protease inhibitors, diagnostics and routine research. However, the current commonly used methods cannot fulfill these requirements because of insufficient sensitivity (e.g. casein gel), complicated manipulations (e.g. trichloroacetic acid precipitation, centrifugation and heating), radioactive hazard (e.g. radio labelled substrates) and expensive equipment required (e.g. fluorescence polarization analyzer), etc. These procedures are usually time-consuming and often do not lend themselves to automation.
According to the present invention, a method of detecting via a solid-phase assay the amount of biological activity and/or the quantity of a biologically active substance is disclosed. The method utilizes the biological activity itself of the substance to provide the method of detection.
In an embodiment of the present invention a method of detecting via a solid-phase assay the amount of biological activity of a biologically active substance utilizing the biological activity is disclosed. A first component is bound to a surface wherein the first component is conjugated to a first indicator. A sample is contacted to the first component. The sample contains a second component having unknown biological activity which is to be measured. The components are in a reaction mixture under conditions such that the biological activity between the first and second component will unbind the first component. After the reaction is complete, the sample is removed. The amount of bound first component remaining is measured. There is a reciprocal relationship between the amount of biological activity and the remaining bound first component.
In a further embodiment of the present invention a method for detecting via a solid-phase assay the amount of an inhibitor of biological activity of a biologically active substance utilizing the inhibition of biological activity is disclosed. A first component is bound to a surface wherein the first component is conjugated to a first indicator. A sample is contacted to the first component. The sample contains a second component having a known amount of a second component having known biological activity and an unknown amount of a third component which is an inhibitor of the second component. The components are allowed to react for a predetermined time under conditions such that the biological activity between the first and second component will unbind the first component and the third component will interfere with the reaction between the first and second component. After the reaction is complete, the sample is removed. The amount of bound first component remaining is determined wherein there is a direct relationship between the remaining bound conjugated first component and amount of the third component in the sample.
The present invention also provides a method of detecting via a solid-phase assay the identity of a biologically active substance utilizing inhibition of the biological activity. A first component conjugated to a first indicator is bound to a surface. A sample is contacted to the first component. The sample contains a second component having a generally known biological activity but is unknown as to its specific activity. The sample also contains a known amount of a third component which is one of a potential inhibitor of the second component. The assay includes a panel of such potential inhibitors. The reaction is allowed to occur under conditions such that the biological activity between the first and second component will unbind the first component and the third component can interfere with the reaction between the first and second component if it is specific for the second component.
After a predetermined time, the sample is removed, generally by washing as is known in the art. The amount of bound first component remaining is determined. If there is a reduction of the amount of the bound first component than the third component did interfere with the reaction between the first and second component thereby identifying the second component. If there is no significant reduction of bound first component than the selected specific inhibitor did not interfere and the second component is not identified. Appropriate standard curves are performed as is known in the art.
The present invention also provides for competitive assays between a first and second component based on the biological activity of the second component.
In a first pair of solid-phase competitive assays the method determines the quantity of a biologically active substance utilizing the biological activity of the substance. A known quantity of a first component which binds to a biologically active second component is bound to the surface of a reaction vessel. A sample or extract containing an unknown quantity of the second component having biological activity is added. The sample also contains a known quantity of the second component coupled to a first indicator. The reaction is run under conditions such that the first and second component will bind due to the biologic activity. After a predetermined time the sample is removed as is known in the art. The amount of second component coupled to a first indicator bound to the first component is then determined. There is a reciprocal relationship between the quantity of the second component coupled to a first indicator bound to the first component and the unknown quantity of the second component having biological activity in the sample. Appropriate standard curves as is known in the art allows quantitation.
In the alternative assay the sample contains an unknown quantity of the first component and a known quantity of the second component coupled to a first indicator. There is a reciprocal relationship between the quantity of the second component coupled to a first indicator bound to the bound first component and the unknown quantity of the first component in the sample.
A second pair of competitive solid-phase assays also provides a method of detecting the quantity of a biologically active substance utilizing the biological activity of the substance. In these assays a known quantity of a second component having biological activity is bound to the surface of a reaction vessel. A sample containing an unknown quantity of the second component and a known quantity of a first component coupled to a first indicator is added. The reaction is run under conditions such that the first and second components will bind due to biologic activity. The sample is removed the sample after the reaction is complete. The amount of first component coupled to a first indicator bound to the bound second component is determined. There is a reciprocal relationship between the quantity of the bound first component coupled to an indicator and the unknown quantity of the second component in the sample.
In the alternative assay of this pair, the sample contains a known quantity of the first component coupled to a first indicator and an unknown quantity of the first component. After the reaction is complete, the amount of first component coupled to a first indicator bound to the bound second component is determined. There is a reciprocal relationship between the quantity of the bound first component coupled to an indicator and the unknown quantity of the first component in the sample.
In these competitive assays the second component can be an enzyme and the first component an inhibitor of the enzyme. Alternatively, the second component is a lectin and the first component is a lectin-binding substance. Further, the assays can use a receptor as the second component and the first component a receptor-binding substance.